Folin-Wu Glucose Test

Principle:
A protein-free blood filtrate is heated with an alkaline copper tartrate solution. The glucose present reduces the copper to cuprous oxide. The cuprous oxide in turn reduces a colorless phosphomolybdic solution to blue phosphomolybdous acid. The intensity of color is measured spectrophotometrically and is proportional to the amount of glucose present .

Sample:
Protein-free filtrate of serum or whole blood. (If whole blood is used, sodium fluoride and potassium oxalate must be used as the anticoagulant.)

Supplies:
Patient Sample: 2 mL Ostwald pipette (1 for each sample tested) 25 ml or 50 mL Erlenmeyer flasks (2 for each sample tested) 1 10 mL serological pipettes 1-1 mL serological pipette 1 5 mL volumetric pipette dH2O 1/12 N sulfuric acid 10% sodium tungstate solution Parafilm filter paper funnels (1 for each sample tested) Normal and abnormal control Patient serum Folin-Wu Procedure: 2- 5mL serological pipettes 3- 2mL volumetric pipettes Alkaline copper tartrate Boiling water bath Ice water bath Phosphomolybdic acid reagent Boiling water bath Ice water bath dH2O Folin-Wu tubes (1 for each sample tested) Parafilm Timer Spectrophotometer Latex gloves Biological Safety Cabinet Lab coat or apron

Calibration: Prepare a standard curve using 4 standard solutions of glucose.

Quality Control: Quality control serum (Dade Moni-Trol I) must be run with each batch of patient tests. The QC is set up according to the procedure. If the QC is out of range, patient results cannot be reported. The QC range is 62-94 mg/dL (normal control) and 240-305 mg/dL (abnormal control).

Procedure: Make a standard curve

1. Using logarithmic graph paper, write the concentration of each standard along the bottom of the paper. 2. Find the % transmittance (%T) along the left side of the graph. 3. Beginning with the 50 mg/dL concentration, find the line that represents the %T which you read on the spectrophotometer. 4. Plot the point where the lines of %T and concentration meet. 5. Repeat steps 3 and 4 for each standard concentration. 6. You should expect to get a fairly straight line when all the points are connected by a line from 0 mg/dL and 100%T to the last of the standards. 7. Use this curve to determine the concentration of the patient and control sera (unknowns). Find the %T reading of the unknown. Move to the right and find the point where the %T line intersects with the standard curve. Draw a vertical line down to the concentration axis. 8. Record the results on chemistry report form. 9. Remember to check your QC values to see if they fall in range.

Prepare a protein-free filtrate

1. Centrifuge blood to separate serum. 2. Measure 2 mL of serum (patient or control) with an Ostwald pipette into a 50 mL Erlenmeyer flask. 3. Add 9 mL dH2O and mix. 4. Add 8 mL of 1/12 N sulfuric acid slowly with a pipette and mix. 5. Add 1 mL of 10% sodium tungstate solution slowly while shaking the flask. 6. Cover with parafilm and shake well. 7. This makes a 1:10 dilution of serum. 8. Shake the mixture again and filter through filter paper. (The filter paper should be large enough so that all the mixture can be filtered at once.) 9. Pour the mixture slowly onto the filter paper so that the paper will be wet above the level of the mixture. 10. Allow the first drops to filter back into the original container before collecting the filtrate.

Folin-Wu procedure
1. Place 2 mL of sample (standard, patient protein-free filtrate, or control protein-free filtrate) into a Folin-Wu glucose tube. Be sure to label all tubes appropriately. 2. Place 2 mL of dH2O into a Folin-Wu glucose tube. This will serve as a blank. (You will have 5 tubes total when preparing the standards and 4 tubes total when preparing the patient and control samples) 3. Add 2 mL of alkaline copper tartrate solution to each tube and mix well by gently shaking tubes. (The surface of the mixture must reach the constricted part of the tube.) 4. Heat immediately in a boiling water bath for 6 minutes. (Set a timer) 5. Cool, without shaking, in a ice water bath for 3 minutes. 6. Add 2 mL of phosphomolybdic acid reagent. 7. When vigorous effervescence has ceased, dilute to the 25 mL mark with dH2O. 8. Cover with parafilm and mix each tube thoroughly by repeated inversion.

9. Within 10 minutes, read the % transmittance on the spectrophotometer at 420 nm. Use the blank to set 100% transmittance.

Results Reporting:
The normal range for glucose using this method is 65-105 mg/dL for serum and 80-120 mg/dL for whole blood. If the patient results are >200 mg/dL, report to physician immediately.

Limitations:
The filtrate from blood specimens must be made up at once unless a sodium fluoride and thymol mixture is used as an anticoagulant. Blood filtrates containing a few drops of toluene may be kept in the refrigerator for 24 hours before analyzing without any appreciable change. Be sure to bring to room temperature before pipetting. All that is reported as blood sugar is not glucose but the sum total of all reducing substances encountered during the determination. Blood from the finger or ear (capillary or arterial blood) gives higher values than venous blood except in the fasting state when they are the same. Directions for heating and cooling the solution must be followed with scrupulous care because variations in temperature or time affect the results significantly. Cuprous compounds produced by sugar in alkaline copper solutions are readily oxidized to the cupric state when exposed to air; therefore, shaking is avoided after the tube is placed in the water bath.

References:
Manual of Clinical Laboratory Methods, Opal Hepler. Charles C. Thomas, Springfield, IL, pp. 265-266, 1975. Chemistry for the Clinical Laboratory, Wilma L. White. C.V. Mosby Co., St. Louis, MO, p. 95, 1976. Medical Laboratory: Student Learning Plan, DISD. DISD, Dallas, TX, pp.541-554, 1979.

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